I. Vacuolar Processing Enzymes (VPEs)
Vacuolar Processing Enzymes (VPEs) are cysteine proteases that cleave a peptide bond at the C-terminal side of asparagine and aspartic acid. VPE was originally discovered as a novel cysteine proteinase responsible for the maturation of seed storage Proteins. Arabidopsis has four VPE genes (alpha VPE, beta VPE, gamma VPE and delta VPE). Hara-Nishimura et al., Current Opinion in Plant Biology 2005, 8:404-408.
In higher plants, proprotein precursors of various vacuolar proteins are converted post-translationally to their respective mature forms by the action of VPEs. The molecular structure of the enzyme was originally reported for castor bean VPE. VPE homologues have been found in plants (soybean, Jack bean, Arabidopsis, vetch and citrus) and animals (Schistosoma mansoni, human and mouse). A VPE precursor is composed of a signal peptide, an N-terminal propeptide, the mature VPE domain and a C-terminal propeptide. A yeast (Saccharomyces cerevisiae) transformant expressing a VPE precursor of castor bean accumulated the mature form in the vacuoles. The mature protein had a vacuolar processing activity; in contrast, the precursor had no activity. Analysis of mutants having no activity suggested that the conversion of the proprotein precursor of VPE into an active form might be mediated self-catalytically. (Hiraiwa et al. FEBS Letters 447, 1999, pp. 213-216).
Programmed cell death (PCD) occurs in animals and plants under various stresses and during development. VPE was identified as an executioner of plant PCD. VPE exhibits enzymatic properties similar to that of a caspase, which is a cysteine protease that mediates the PCD pathway in animals, although there is limited sequence identity between the two enzymes. VPE was reported to have caspase-1 activity (Hatsugai et al., SCIENCE VOL 305 6 Aug. 2004). VPE and caspase-1 share several structural properties: the catalytic dyads and three amino acids forming the substrate pockets (Asp pocket) are conserved between VPE and caspase-1. In contrast to such similarities, VPE is localized in the vacuoles, while caspases are localized in the cytosol. VPE functions as a key molecule of plant PCD through disrupting the vacuole in pathogenesis and development. Hatsugai et al., Apoptosis 2006; 11: 905-911. VPE gamma (VPEg), was reported to be induced during senescence, a form of PCD (see Rojo et al., 2004, Current Biology, Vol. 14, pp 1897-1906).
II. CCA1
MYB proteins are a superfamily of transcription factors that play regulatory roles in developmental processes and defence responses in plants. Expression analysis revealed that the expression for most of the Arabidopsis MYB genes were responsive to one or multiple types of hormone and stress treatments (Yanhui et al., Plant Mol. Biol. 60, 107-124, 2006). A phylogenetic comparison of the members of this superfamily in Arabidopsis and rice suggested that the Arabidopsis MYB superfamily underwent a rapid expansion after its divergence from monocots but before its divergence from other dicots (Yanhui et al., 2006). MYB proteins typically comprise a structurally conserved DNA-binding domain, the MYB domain. They are involved in the cell cycle, regulation of meristem formation, control of cellular differentiation and in the regulation of secondary metabolism. MYB domain transcription factors constitute one of the largest family of transcription factors in plants (at least 130 in Arabidopsis thaliana), but with little sequence conservation outside of the MYB domain. They have therefore been clustered into subgroups based on conserved motifs identified outside of the MYB coding region (Jiang et al. (2004) Genome Biology 5:R46). Different categories of MYB proteins can be identified depending on the number of imperfect repeats of the MYB domain they contain, grouped into 3 families: R2R3-MYB family, R1R2R3-MYB family and the MYB-related family. The R2R3-MYB family comprises the largest number of MYB proteins and is divided into five subfamilies (Yanhui et al. 2006): CCA1-like, CPC-like, TBP-like, 1-box-binding-like and R-R-type MYB proteins. Of these, the CCA1-like subfamily is the largest, and members of this subfamily contain the conserved motif SHAQK or MYADN in the MYB repeat.
The circadian clock controls various physiological and molecular processes in higher organisms. In plants, these processes include leaf movement, stomata opening, and expression of a large number of genes. In Arabidopsis thaliana, a number of clock-associated protein components have been identified. Among them, CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1)/LHY (LATE ELONGATED HYPOCOTYL) and TOC1 (TIMING OF CAB EXPRESSION 1) are believed to be the essential components of the central oscillator. CCA1 and LHY are homologous and partially redundant Myb-related DNA-binding proteins, whereas TOC1 is a member of a small family of proteins, designated as PSEUDO-RESPONSE REGULATOR. It is also believed that these two different types of clock components form an autoregulatory positive/negative feedback loop at the levels of transcription/translation that generates intrinsic rhythms (Nakamichi et al., Plant Cell Physiol. 46, 686-689, 2005). It was reported that constitutive expression of the CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) gene in Arabidopsis plants (CCA1-ox) results in loss of circadian rhythmicity (Green et al., Plant Physiol. 129, 576-584, 2002). These CCA1-ox plants retain the ability to respond to diurnal changes in light. Thus, transcript levels of several circadian-regulated genes, as well as CCA1 itself and the closely related LHY, oscillate robustly if CCA1-ox plants are grown under diurnal conditions. However, in contrast with wild-type plants in which transcript levels change in anticipation of the dark/light transitions, the CCA1-ox plants have lost the ability to anticipate this daily change in their environment. CCA1-ox plants flowered later, especially under long-day conditions, and were less viable under very short-day conditions than their wild-type counterparts. In addition, it was demonstrated that two other circadian rhythm mutants, LHY-ox and elf3, have low-viability phenotypes.
WO2003013228 and US2004019927 describe that CCA1 overexpressing plants were late bolting, showed increased biomass (increased leaf number and size), and were darker green in vegetative and reproductive tissues. It was suggested that CCA1 could be useful in increasing chlorophyll content allowing more growth and productivity in conditions of low light.
Furthermore, it was stated that use of CCA1 to prevent flowering could help maximize vegetative yields and prevent escape of genetically modified organism (GMO) pollen (US2004045049). So far, there are no reports showing that increased CCA1 expression results increased seed yield, on the contrary, CCA1-overexpressors flowered later, especially under long-day conditions, and were less viable under very short-day conditions than their wild-type counterparts (Green et al., 2002).
III. SAP
The SAP domain (named after SAF-A/B, Acinus and PIAS) is a DNA binding domain that forms a helix-extended-helix structure. Proteins with a SAP domain (also named SAF box) have been identified in yeast, mammals and plants. The SAP domain is composed of 35 amino acids residues and comprises two amphipathic helices separated by a glycine-containing region. Some positions in this domain are enriched with positively charged amino acids (R, K) which are thought to contact the DNA backbone. The SAP domain reportedly forms a helix-extended-helix (HEH) structure and that some prokaryotic proteins, such as transcription terminator RHO protein, are also predicted to contain SAP domains. Chen et al., 2003 (Plant Molecular Biology 52: 579-590) report that SAP domain-containing proteins have been implicated in various functions related to their interactions with DNA and/or RNA. SAP proteins have been implicated in nuclear architecture and/or RNA metabolism. For example, human scaffold attachment factor A (SAF-A) is an abundant component of the nuclear scaffold (nuclear matrix) and is also present in heterogeneous nuclear ribonucleoprotein complexes, which have been implicated in nuclear organization and RNA processing. Acinus is a caspase-3-activated protein required for apoptotic chromatin condensation. Members of the PIAS proteins family combining SAP domains and MIZ Zn-finger motifs are the protein inhibitors of activated STATs (signal transducer and activator of transcription). Yeast Tho1p protein, another SAP-containing protein, plays a role in regulating elongation of transcription by RNA polymerase II. Chen et al., 2003, describe the cloning and characterization of a rice gene, OsBP-73, encoding a 375 amino acid protein with a SAP-like domain. The authors report that Northern blot analysis demonstrated that OsBP-73 is weakly expressed in root, leaf and immature seed. They also examined OsBP-73 gene expression by histochemical studies of transgenic rice plants carrying an OsBP-73 5_/GUS reporter gene. The reporter gene was found to be mainly expressed in the tissues with high cell division activities, such as root tip, stem node, panicle and immature seed. They further report that genetic interference of OsBP-73 gene expression by double-stranded RNA inhibits the whole plant growth but does not affect the passage from the juvenile to adult phase. They suggest that OsBP-73 may play an important role in the regulation of cell proliferation.
IV. SYPF1
SYPF1 is a novel transcription factor useful in enhancing yield-related traits in plants.
Transcription factors are usually defined as proteins that show sequence-specific DNA binding and that are capable of activating and/or repressing transcription. The Arabidopsis genome codes for at least 1533 transcriptional regulators, which account for ˜5.9% of its estimated total number of genes. About 45% of these transcription factors are reported to be from families specific to plants (Riechmann et al., 2000 (Science Vol. 290, 2105-2109)).
SYPF1, according to the PRODOM database, was found to share some similarity to tumor related At4g18650; TGA1 bzip activator coil coil. Miao et al., 1994 (Plant Mol. Biol. April 25(1): 1-11) report that TGA1a is a well-characterized transcription factor that may mediate the root-specific and auxin-responsive expression of some plant genes.
V. Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) Activase (RCA) Polypeptide
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo, EC 4.1.1.39 [EC]) is the most abundant and one of the most important enzymes on earth. It catalyses the first and rate-limiting step in photosynthetic carbon fixation, the irreversible carboxylation of ribulose-1,5-bisphosphate and CO2 to form two 3-phosphoglyceric acid molecules. However, the rate of the reaction is extremely slow, and RuBisCo must be activated and carbamylated to become catalytically competent. Activation is achieved by RuBisCo activase (RCA), which can remove inhibitors from RuBisCo's catalytic sites, alter the conformation, and activate RuBisCo in vivo in an ATP-dependent manner (Andrews et al., 1995).
RCA is a nuclear-encoded chloroplast protein that is a member of the AAA+ family (ATPases associated with diverse cellular activities) based on sequence and structural homologies, whose members participate in macromolecular complexes that perform diverse chaperone-like functions. Consistent with the ATPase activity of RCA, a P loop (or Walker A; Walker et al., (1982) EMBO J. 1982; 1:945-951) triphosphate-binding loop consensus sequence GXXXGK(S/T), for nucleotide binding, is identified within the RCA polypeptide sequence. Several other critical amino acid residues necessary for RCA interaction with and activation of RuBisCo have also been identified (for review: Portis (2003) Photosynthesis Research 75: 11-27).
RCA consists in most plants of two isoforms, of 45-46 kDa (or alpha form) and of 41-43 kDa (or beta form), arising from a single gene via alternative splicing (for review, see Pots (2003) Photosynthesis Research 75: 11-27). The two forms differ only at the carboxy terminus, the longer form comprising two cysteine residues involved in light-dependent redox regulation (mediated by thioredoxin-f). In contrast with most plants, a single polypeptide (without the carboxy terminal extension) has been found in the green alga Chlamydomonas reinhardtii, which also comprises a chloroplast transit peptide at the amino terminus of the polypeptide (Roesler & Ogren (1990) Plant Physiol 94(4):1837-1841).
Mutant Arabidopsis plants lacking RCA activity (named rca-; Somerville et al. (1982) Plant Physiol 70: 381-387) or transgenic plants having a very low level of RCA activity cannot survive at atmospheric CO2 levels (Flaveria bidentis; von Caemmerer et al. (2005) Plant Physiol 137(2):747-55), and those expressing reduced levels exhibit reduced rates of photosynthesis and growth (in Arabidopsis, Eckhardt et al. (1997) Plant Physiol 113: 575-586; in tobacco, Mate et al. (1996) Planta 198: 604-613). However large reductions in RCA activity levels are required before steady-state photosynthesis is noticeably affected, at normal temperatures (for example, Arabidopsis, rice (Jin et al. (2006) Ann Bot (Lond) 97(5):739-44), tobacco (He et al. (1997) Plant Physiol 115(4):1569-80; Hammond et al. (1998) Plant J 14: 101-110).
Arabidopsis mutants lacking RCA (rca-) were transformed (complemented) with the alpha RCA isoform, a mutated alpha RCA isoform (the two Cys residues involved in redox regulation are mutated in Ala residues), the beta RCA isoform, or both RCA isoforms restored the ability of the plants to grow under normal levels of CO2 (Zhang et al. (2002) PNAS USA 99(5): 3330-3334). Plants expressing only the beta RCA isoform (and thus not light-dependent redox-regulated) or expressing only the mutated alpha RCA isoform (redox insensitive) were incapable of down-regulating RuBisCo under limiting light conditions.
Patent application US2006/0272044 relates to methods (by gene shuffling) for obtaining isolated polynucleotides sequences encoding RCA polypeptides having enhanced activity.
Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding a YEP selected from a Vacuolar Processing Enzyme (VPE), a CCA1-like polypeptide, a SAP-like polypeptide, a Seed Yield Promoting Factor 1 (SYPF1) polypeptide and a Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activase (RCA) polypeptide gives plants having enhanced yield-related traits relative to control plants.